Many breast tumors are estrogen-dependent, meaning that they require estrogens for growth. For several years, a popular and successful breast tumor treatment strategy has employed antiestrogens. Antiestrogens (such as tamoxifen) are a class of compounds which inhibit estrogens from eliciting their full response in target tissues. Tamoxifen has also been used as a prophylatic drug for women with a high risk for breast cancer. However, many antiestrogens do not act as strict antagonists, but rather also act as partial agonists on the estrogen receptor (ER). This partial agonist activity has proven to be a `double-edged sword`. While the partial agonist activity has been shown to produce beneficial effects such as reducing serum cholesterol and preventing osteoporosis, it has recently been implicated by a number of independent studies as promoting increased endometrial tumor formation. (Endometrial cells consist of stromal and luminal epithelial cells, which in addition to smooth muscle myometrial cells, comprise the major cell types of the uterus.) For instance, findings from the National Surgical Adjuvant Breast and Bowel Project concluded that "Risk of endometrial cancer increases following tamoxifen treatment for breast cancer; however, net benefit greatly outweighs risk" (Fisher et al. (1994)). The increased incidence of endometrial cancer after adjuvant tamoxifen therapy for breast cancer was recently confirmed in a study of 87,323 women with breast cancer as well as others (Rutqvist et al. (1995), Fornander et al. (1989), Mouridsen et al. (1988), Ryden et al. (1986)). The partial agonistic or "estrogen-like" activity of tamoxifen in the human uterus and the effects of tamoxifen on increased incidence of endometrial cancer is consistent with previous studies in laboratory animal and cell culture models. For instance, tamoxifen induced uterine hyperplasia in most animal models and tumor growth in athymic nude mice bearing transplanted estrogen-responsive endometrial tumors.
Thus, the prior art method treating estrogen-dependent breast tumors results in a significant increase in the incidence of endometrial tumors. This increased incidence of endometrial tumors may be due to estrogen-like activity of tamoxifen in the uterus.
More recently, another area of research has focused on the antiestrongenic activity of aryl hydrocarbon receptor (AhR) agonists using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a model compound. TCDD and related compounds inhibit mammary tumor growth in rodent models and 17.beta.-estradiol (E2)-induced responses in the rodent uterus and human breast cancer cells. While TCDD was demonstrated to be an effective antiestrogen in rats, mice, and MCF-7 human breast cancer cell lines in culture, it proved to be too hepatocarcinogenic for use as a therapeutic antiestrogenic. Previous studies have demonstrated that TCDD induces toxic and biochemical responses in the rat; one such biochemical response, instruction of CYP1A1 dependent aryl (hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase (EROD) activities in the liver correlates with the toxicity of TCDD.
A series of 6-alkyl-1,3,8-trichlorodibenzofurans (triCDFs) were originally synthesized for investigating their activities as partial AhR antagonists. The 6-alkyl-1,3,8-triCDFs are of the formula: ##STR1## where R.sub.1, R.sub.3, and R.sub.8 are chlorine, R.sub.6 is a linear of branched alkyl group of one to four carbons, and R.sub.2, R.sub.4, R.sub.7, and R.sub.9 are hydrogen. 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF) was used as a prototype for this series of compounds.
Initial studies showed that 6-MCDF bound to the Ah receptor but was a weak inducer of CYP1A1 and exhibited minimal toxicity. Moreover, 6-MCDF inhibiter TCDD induced toxic responses and was characterized as an Ah receptor antagonist, (B. Astroff et al. (1988); M. Harris et al. (1989); R. Bannister et al. (1989); C. Yao et al. (1989)). However, 6-MCDF did not inhibit TCDD-induced antiestrogenic activity; 6-MCDF was also an antiestrogen in the rat uterus. (B. Astroff and Safe (1991)). In 1988, Astroff and Safe reported that both TCDD and 6-MCDF caused a dose-dependent decrease in nuclear and cytosolic ER and progesterone receptor (PR) binding in 21 to 25 day-old female Sprague-Dawley rat uterus. Moreover, 6-MCDF was only 300 to 570 times less active than TCDD as an antiestrogen while it was &gt;157,000 times less potent than TCDD as an inducer of hepatic CYP1A1 in the same animals (CYP1A1 is a surrogate for toxic potency). Subsequent studies showed that 6-MCDF and related compounds inhibited E2-indued hypertrophy, peroxidase activity, cytosolic ER and PR binding, and EGF receptor and c-fos mRNA levels in the rat uterus. Thus, it was apparent that 6-MCDF was capable of the beneficial antiestrogenicity effect without the toxic side effects of TCDD.
The in vivo antiestrogenic activity of a series of alkyl-substituted polychlorinated dibenzofurans (PCDFs) has been investigated in the immature female Sprague-Dawley rat uterus (B. Astroff et al. (1991); R. Dickerson et al. (1995)). The compounds utilized in this study contain two, three, or four lateral substituents and include: 6-MCDF, 6-ethyl-1,3,8-triCDF, 6-n-propyl-1,3,8-triCDF, 6-i-propyl-1,3,8-triCDF, 6-t-butyl-1,3,8-triCDF, 8-MCDF (two lateral substituents); 6-methyl-2,3,8-triCDF, 6-methyl-2,3,4,8-tetraCDF, 8-methyl-1,3,7-triCDF, and 8-methyl-1,2,4,7-tetraCDF (three lateral substituents); 8-methyl-2,3,7-triCDF, 8-methyl-2,3,4,7-tetraCDF (four lateral substituents). Two additional compounds, 8-methyl-2,3,7-trichlorodibenzo-p-dioxin and 8-methyl-2,3,7,-tribromodibenzo-p-dioxin (four lateral substituents), were also investigated. All alkyl-substituted compounds inhibited estrogen-induced uterine wet weight increase and cytosolic and nuclear PR and ER binding. Quantitative structure-antiestrogenicity relationships were determined using 6-i-propyl-1,3,8-triCDF, 6-methyl-2,3,4,8-tetraCDF, and 8-methyl-2,3,4,7-tetraCDF as representative congeners containing two, three and four lateral substituents, respectively. The ED.sub.50 values (ED.sub.50 is defined as the dose which produces 50% of a maximal response) for antiestrogenicity were similar for the three compounds; however the ED.sub.50 values for induction of hepatic CYP1A1-dependent activity were 73,600 (estimated), 8.52, and 5.31 mmol/kg for 6-i-propyl-1,3,8,-triCDF, 6-methyl-2,3,4,8-tetraCDF, and 8-methyl-2,3,4,7-tetraCDF, respectively. Based on results of previous studies, CYP1A1 can be used as a surrogate for toxic potency in the rat; therefore, high ED.sub.50 (CYP1A1 induction)/ED.sub.50 (antiestrogenicity) ratios are indicative of low toxicity and high antiestrogenic potency. The ratio was 13,990-17,100 for 6-i-propyl-1,3,8-triCDF, whereas corresponding ratios for the compounds with three and four lateral substituents varied from 0.64-3.34. These data suggested that alternate 1,3,6,8-substituted alkyl PCDFs are useful structural models for developing new AhR-mediated antiestrogens for treatment of breast cancer. Data regarding the antiestrogenic effects of 1,3,6,8- or 2,4,6,8-alternate substituted alkyl dibenzofurans have been presented in U.S. Pat. No. 5,516,790 and are hereby incorporated by reference in their entirety.
The in vivo antitumorigenic activity of 6-MCDF, 8-MCDF (8-methyl-1,3,6-triCDF) and 6-cyclohexyl-1,3,8-triCDF (6-CHDF) were investigated in the DMBA rat mammary tumor model (McDougal et al., Inhibition of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor growth by aryl hydrocarbon receptor agonists. Cancer Letters 120:53-63, 1997. At doses of 5, 10 or 25 mg/kg/wk, 6- and 8-MCDF significantly inhibited mammary tumor growth, and at the 5 mg/kg/wk dose, &gt;50% growth inhibition was observed for both isomers. In contrast, 6-CHDF was inactive at the 5 mg/kg/wk dose, and the structure-antitumorigenicity relationships (6-/8-MCDF&gt;&gt;6-CHDF) correlated with structure-antiestrogenicity (rat uterus) studies and the relative binding affinities of these compounds for the AhR. The antitumorigenic activity of 6- or 8-MCDF in the mammary was not accompanied by any significant changes in liver/body weight ratios, liver morphology or induction of hepatic CYP1A1-dependent activity which is one of the most sensitive indicators of exposure to AhR agonists. RT-PCR and Western blot analysis of mammary tumor mRNA and protein extracts, respectively, confirmed the presence of the AhR suggesting that AhR-mediated signaling pathways are functional in rat mammary tumors, but simply not activated by 6- or 8-MCDF.
This invention is based on utilizing a well-described antiestrogenic drug (tamoxifen) in combination with alkyl PCDF's. The alkyl PCDFs can interact with tamoxifen (actively) and in combined treatment over the effective dose of tamoxifen required for treatment of breast cancer and at the same time provide protection from tamoxifen-induced endometrial cancer.
While not intending to be bound by theory, it is believed that tamoxifen and alkyl PCDFs function via two different mechanisms. AhR is believed to decrease the ER by an unknown mechanism (presumably posttranscriptional). It is believed that alkyl PCDFs work through the AhR which cross-talks with the ER. Tamoxifen is believed to bind the ER and block ER action in the breast. 6-MCDF is believed to act through the AhR and therefore bind to the receptor. 6-MCDF is believed to be an AhR antagonist for the toxic responses (i.e., it inhibits TCDD toxicity in co-treatment studies) but is believed to act as an Ah receptor agonist for antiestrogenicity (i.e., acts like TCDD).
The prior art method of treating estrogen-dependent breast tumors frequently involves the administration of tamoxifen. As mentioned above, tamoxifen therapy has been shown to be associated with a significantly elevated incidence for endometrial tumor formation in several independent trials (Fomander et al. (1989), Rutqvist et al. (1995), Mouridsen et al. (1988), Ryden et al. (1986), Fisher et al. (1994)). Some studies have suggested that the incidence of endometrial tumor formation may be reduced with lower tamoxifen doses. The Stockholm trial demonstrated an approximately 6 fold higher risk for endometrial tumors with a treatment protocol of 40 mg/day for 2 years (Fomander et al. (1989), Rutqvist et al. (1995)), while the Danish and South-Swedish trials demonstrated only 3.3 and 2.0 fold higher risks, respectively. In the latter two trials, tamoxifen was administered at 30 mg/day for one year. In Scottish and Manchester England trials in which tamoxifen was administered at 20 mg/day, there appeared to be no increased risk for endometrial tumors (Stewart et al. (1989), Stewart (1992), Ribeiro et al. (1992)). However, a third study involving 2843 patients demonstrated a 7.5 fold increased risk for endometrial tumor formation with the same dose (20 mg/day) of tamoxifen (Fisher et al. (1994)). In light of the smaller population sizes of the former studies, the results of the Scottish and Manchester England trials should be interpreted cautiously. Thus, it appears clear from the preponderance of studies that tamoxifen therapy results in significantly increased risks for endometrial tumor formation. However, it is presently unclear from existing trials whether a reduction in tamoxifen dose results in a reduced risk of endometrial tumor formation.
Although there is no clear indication that reducing tamoxifen dose correspondingly reduces the risk for endometrial tumor formation, it should be kept in mind that large population based trials have not been conducted with tamoxifen doses less than 20 mg/day. Data from animal and cell culture models, however, do suggest a dose-related uterotropic effect for tamoxifen and this is consistent with pharmacological principles. For example, tamoxifen produced significant dose dependent increases in rat uterine wet weight which correlated with increased drug concentration. Therefore, it follows that similar reductions in tamoxifen dose might also result in diminished endometrial tumor formation.
What is needed is a method for reducing the necessary concentration of tamoxifen without reducing efficacy. Ideally, such a method should maintain the beneficial effects of tamoxifen (such as on maintaining bone mass) and minimize the tumorogenic effects of tamoxifen in the uterus.